Methods of tagging rna molecules associated with epigenetically modified chromatin in living cells

Abstract

A method of tagging RNA molecules involved in an epigenetically modified chromatin in real-time in living cells is provided. The method involves the introduction of an epigenetic reader module protein, an engineered ascorbate peroxidase, and a biotin-aniline probe into a living cell. Subsequently, the biotin-aniline probe is oxidized by the engineered ascorbate peroxidase, resulting in the formation of a covalent bond between the probe and an RNA molecule within the epigenetically modified chromatin, thereby generating a biotin-tagged RNA molecule. Finally, the biotin-tagged RNA molecule is purified for subsequent sequencing analysis.

1 . A method of tagging RNA molecules involved in an epigenetically modified chromatin in real-time in living cells, comprising:
 introducing an epigenetic reader module protein, an engineered ascorbate peroxidase and biotin-aniline probe into a living cell;   oxidizing the biotin-aniline probe by the engineered ascorbate peroxidase and forming a covalent bond between the biotin-aniline probe and an RNA molecule of an epigenetically modified chromatin to obtain a biotin-tagged RNA molecule; and   purifying the biotin-tagged RNA molecule for further sequencing.   
     
     
         2 . The method of  claim 1 , wherein the epigenetic reader module protein locates a location of the epigenetically modified chromatin and drags the engineered ascorbate peroxidase to the location. 
     
     
         3 . The method of  claim 1 , wherein the oxidation of the biotin-aniline probe occurs when the engineered ascorbate peroxidase is exposed to H 2 O 2 . 
     
     
         4 . The method of  claim 1 , wherein the epigenetic reader module protein is an evolutionarily conserved protein derived from natural proteins. 
     
     
         5 . The method of  claim 4 , wherein the epigenetic reader module protein comprises chromodomain from CBX7 or Drosophila Polycomb (dPC) for H3K27me3 (trimethyl-histone H3 lysine 27), chromodomain from CBX1 for H3K9me3 (trimethyl-histone H3 lysine 9), and PHD domain from TAF3 for H3K4me3. 
     
     
         6 . The method of  claim 4 , wherein the epigenetic reader module is further fused with a plurality of a repetitive peptide epitope and the engineered ascorbate peroxidase is fused with a single-chain variable fragment (scFv). 
     
     
         7 . The method of  claim 6 , wherein the repetitive peptide epitope is recognizable by the scFV for recruiting the engineered ascorbate peroxidase. 
     
     
         8 . The method of  claim 7 , wherein the repetitive peptide epitope has an amino acid sequence of SEQ ID NO:01 and the scFV has an amino acid sequence of SEQ ID NO:02. 
     
     
         9 . A kit for tagging RNA molecules involved in an epigenetically modified chromatin in real-time in living cells, comprising:
 an epigenetic reader module protein;   an engineered ascorbate peroxidase; and   a biotin-aniline probe.   
     
     
         10 . The kit of  claim 9 , wherein the components of the kit are directly delivered into a living cell. 
     
     
         11 . The kit of  claim 9 , wherein the engineered ascorbate peroxidase oxidizes the biotin-aniline probe when the engineered ascorbate peroxidase is exposed to H 2 O 2 . 
     
     
         12 . The kit of  claim 11 , wherein the oxidized biotin-aniline probe forms a covalent bond to an RNA molecule of an epigenetically modified chromatin to generate a biotin-tagged RNA molecule. 
     
     
         13 . The kit of  claim 12 , wherein the biotin-tagged RNA molecule is further purified and enriched. 
     
     
         14 . The kit of  claim 9 , wherein the epigenetic reader module protein locates a location of the epigenetically modified chromatin and drags the engineered ascorbate peroxidase to the location. 
     
     
         15 . The kit of  claim 9 , wherein the epigenetic reader module protein is an evolutionarily conserved protein derived from natural proteins. 
     
     
         16 . The kit of  claim 9 , wherein the epigenetic reader module protein comprises chromodomain from CBX7 or dPC for H3K27me3, chromodomain from CBX1 for H3K9me3, and PHD domain from TAF3 for H3K4me3. 
     
     
         17 . The kit of  claim 9 , wherein the epigenetic reader modules is further fused with a plurality of a repetitive peptide epitope and the engineered ascorbate peroxidase is fused with a scFv. 
     
     
         18 . The kit of  claim 17 , wherein the repetitive peptide epitope is recognizable by the scFV for recruiting the engineered ascorbate peroxidase. 
     
     
         19 . The kit of  claim 18 , wherein the repetitive peptide epitope has an amino acid sequence of SEQ ID NO:01 and the scFV has an amino acid sequence of SEQ ID NO:02.